This proposal addresses several fundamental biochemical questions about two of the major membrane-bound enzymes found in catecholamine secretory granules - dopamine-Beta-hydroxylase (DBH) and cytochrome b561. Our work during the previous grant period has resulted in the purification and reconstitution of these two enzymes into phospholipid vesicles. New hypotheses about the molecular structure of DBH and also about the functional interaction of DBH with cytochrome b561 have been developed. The biochemistry of DBH is especially interesting because it is found in both soluble and membrane-bound forms and is secreted by the adrenal medulla and sympathetic neurons. The first objective of the program is to follow through on our discovery that DBH has non-identical subunits and identify the molecular structure of the different subunits of DBH and to elucidate the effect of subunit interaction on enzyme activity. This is important for elucidation of the cellular processing and functional differentiation of secreted and membrane-bound forms of DBH. The methods used will be peptide mapping of the isolated DBH subunits, HPLC characterization of subunit association in dissociating conditions, and kinetic analysis of isologous and heterologous tetrameric DBH in both soluble and membrane-reconstituted forms. This knowledge is also important to the clinical assay of DBH activity in serum, lymph, and cerebral spinal fluid because interpretation of such clinical data depends on the molecular origin and association of DBH subunits found in body fluids. The second major objective of the program is to elucidate the role of cytochrome b561 as an intermediate in the regeneration of reducing equivalents for DBH activity. This will involve use of both chromaffin granule ghost membranes and reconstituted phospholipid vesicles in experiments designed to show the role of the cytochrome in reduction of either semidehydroascorbate or dehydroascorbate. This hypothetical role is based on our observation that the redox equilibrium between ascorbate and the cytochrome is uniquely poised to take advantage of the normal transmembrane pH gradient to act as an electron shuttle for the ascorbate system. The third objective is to follow through on our finding that the cytochrome and DBH associate in vitro and thus may be associated in the native membrane. We plan to develop a new technique using antibody extraction of these proteins from lipid diluted membranes as a novel way to assay membrane protein associations.